1. Field of the Invention
This invention relates to novel multibinding compounds (agents) that are 3 adrenergic receptor agonists and pharmaceutical compositions comprising such compounds. Accordingly, the multibinding compounds and pharmaceutical compositions of this invention are useful in the treatment and prevention of metabolic disorders such as obesity, diabetes, and the like.
References
The following publications are cited in this application as superscript numbers:
1 Hardman, J. G., et al. xe2x80x9cThe Pharmnacological Basis of Therapeuticsxe2x80x9d, McGraw-Hill, New York, (1996)
2 Strosberg, A. D. xe2x80x9cStructure, Function, and Regulation of Adrenergic Receptorsxe2x80x9d Protein Sci. 2, 1198-1209 (1993).
3 Beck-Sickinger, A. G. xe2x80x9cStructure Characterization and Binding Sites of G-Protein-coupled Receptorsxe2x80x9d DDT, 1, 502-513, (1996).
4 Hein, L. and Kobilka, B. K. xe2x80x9cAdrenergic Receptor Signal Transduction and Regulationxe2x80x9d Neuropharmacol, 34, 357-366, (1995).
5 Strosberg, A. D. and Pietri-Rouxel, F. xe2x80x9cFunction, and Regulation of xcex23 Adrenoceptorxe2x80x9d TiPS, 17, 373-381, (1996).
6 Kurscheid, T. et al. xe2x80x9cThe cost implications of obesity of health care and societyxe2x80x9d Intl. J. of Obesity, 22 (suppl. 1):S3, (1998).
7 Weiser, et al. xe2x80x9cPharmacologic approach to obesityxe2x80x9d. J. Clin. Pharmacol. 37:453, (1997).
All of the above publications are herein incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference in its entirety.
2. State of the Art
A receptor is a biological structure with one or more binding domains that reversibly complexes with one or more ligands, where that complexation has biological consequences. Receptors can exist entirely outside the cell (extracellular receptors), within the cell membrane (but presenting sections of the receptor to the extracellular milieu and cytosol), or entirely within the cell (intracellular receptors). They may also function independently of a cell (e.g., clot formation). Receptors within the cell membrane allow a cell to communicate with the space outside of its boundaries (i.e., signaling) as well as to function in the transport of molecules and ions into and out of the cell.
A ligand is a binding partner for a specific receptor or family of receptors. A ligand may be the endogenous ligand for the receptor or alternatively may be a synthetic ligand for the receptor such as a drug, a drug candidate or a pharmacological tool.
The super family of seven transmembrane proteins (7-TMs), also called G-protein coupled receptors (GPCRs), represents one of the most significant classes of membrane bound receptors that communicate changes that occur outside of the cell""s boundaries to its interior, triggering a cellular response when appropriate. The G-proteins, when activated, affect a wide range of downstream effector systems both positively and negatively (e.g., ion channels, protein kinase cascades, transcription, transmigration of adhesion proteins, and the like).
Adrenergic receptors (AR) are members of the G-protein coupled receptors that are composed of a family of three receptor sub-types: xcex11 (A, B, D) xcex12 (A, B, C), and xcex2(1, 2, 3).1-5 These receptors are expressed in tissues of various systems and organs of mammals and the proportions of the xcex1 and the xcex2 receptors are tissue dependant. For example, xcex21 is found in cardiac tissue, xcex22 is found in the uterus, skeletal muscle, and lungs5 and xcex23 is predominantly found in adipose tissue5.
It has been established that obesity is the main cause of non-insulin dependent diabetes (NIDDM) and an important factor for cardiovascular disease6.7. It has been shown that treatment with xcex23-AR agonists: 1) reduces diet-induced obesity in mice; 2) leads to reduction of weight in adult dogs; and 3) regulates lipolysis in human adipocytes expressing xcex23-AR, in vitro9.
Currently, a number of xcex23-AR agonists such as BRL 26830A, BRL 35135, Ro 16-8174, Ro 40-2148 and CL 316,24322 are development for the treatment of obesity. Unfortunately, the half -lives of these drugs are short and their bioavailabilty is poor7. Furthermore, they suffer from adverse side effects including cardiovascular abnormalities, tremors, insomnia, dizziness, and elevated systolic blood pressure. Accordingly, there is a need for long acting, xcex23-AR selective drugs that are efficacious and lack unpleasant side effects.
The multibinding compounds of the present invention fulfill this need.
This invention is directed to novel multibinding compounds (agents) that are xcex23 adrenergic receptor agonists and are therefore useful in the treatment and prevention of diseases related to metabolic disorders such as obesity, diabetes, and the like.
Accordingly, in one of its composition aspects, this invention provides a multibinding compound of Formula (I): 
wherein:
p is an integer of from 2 to 10;
q is an integer of from 1 to 20,
each ligand, L, is independently of each other:
(i) a compound of formula (a): 
wherein:
Ar1 is selected from the group consisting of aryl, heteroaryl, cycloalkyl, substituted cycloalkyl, and heterocyclyl wherein each of said Ar1 substituent optionally links the ligand to a linker via a covalent bond;
R1 and R2 are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, and a covalent bond that links the ligand to a linker;
R3 is selected from the group consisting of hydrogen, alkyl, and a covalent bond that links the ligand to a linker; or
(ii) a compound of formula (b): 
wherein:
Ar2 is selected from the group consisting of aryl, heteroaryl, cycloalkyl, substituted cycloalkyl, and heterocyclyl wherein each of said Ar2 substituent optionally links the ligand to a linker via a covalent bond;
R4 and R5 are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, and a covalent bond that links the ligand to a linker;
W is a covalent bond, alkyl or substituted alkyl where one or more of the carbon atoms in said alkyl or substituted alkyl group is optionally replaced by one or more heteroatom selected from xe2x80x94Oxe2x80x94, S(O)nxe2x80x94 (where n is an integer from 0 to 2), or xe2x80x94NR4xe2x80x94 (where R4 is hydrogen, or alkyl); and
Ar3 is selected from the group consisting of aryl, heteroaryl, cycloalkyl, substituted cycloalkyl, and heterocycle wherein each of said Ar substituent optionally links the ligand to a linker via a covalent bond;
each linker, X, in the multibinding compound of Formula (I) independently has the formula:
xe2x80x94Xaxe2x80x94Zxe2x80x94(Yaxe2x80x94Z)mxe2x80x94Xaxe2x80x94
xe2x80x83wherein:
m is an integer of from 0 to 20;
Xa at each separate occurrence is selected from the group consisting of xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94NRxe2x80x94, xe2x80x94C(O)xe2x80x94, xe2x80x94C(O)Oxe2x80x94, xe2x80x94OC(O)xe2x80x94, xe2x80x94C(O)NRxe2x80x94, xe2x80x94NRC(O)xe2x80x94, C(S), xe2x80x94C(S)Oxe2x80x94, xe2x80x94C(S)NRxe2x80x94, xe2x80x94NRC(S)xe2x80x94, or a covalent bond where R is as defined below;
Z at each separate occurrence is selected from the group consisting of alkylene, substituted alkylene, cycloalkylene, substituted cylcoalkylene, alkenylene, substituted alkenylene, alkynylene, substituted alkynylene, cycloalkenylene, substituted cycloalkenylene, arylene, heteroarylene, heterocyclene, or a covalent bond;
each ya at each separate occurrence is selected from the group consisting of xe2x80x94Oxe2x80x94, xe2x80x94C(O)xe2x80x94, xe2x80x94OC(O)xe2x80x94, xe2x80x94C(O)Oxe2x80x94, xe2x80x94NRxe2x80x94, xe2x80x94S(O)nxe2x80x94, xe2x80x94C(O)NRxe2x80x2xe2x80x94, xe2x80x94NRxe2x80x2C(O)xe2x80x94, xe2x80x94NRxe2x80x2C(O)NRxe2x80x2xe2x80x94, xe2x80x94NRxe2x80x2C(S)NRxe2x80x2xe2x80x94, xe2x80x94C(xe2x95x90NRxe2x80x2)xe2x80x94NRxe2x80x2xe2x80x94, xe2x80x94NRxe2x80x2xe2x80x94C(xe2x95x90NRxe2x80x2)xe2x80x94, xe2x80x94OC(O)xe2x80x94NRxe2x80x2xe2x80x94, xe2x80x94NRxe2x80x2xe2x80x94C(O)xe2x80x94Oxe2x80x94, xe2x80x94Nxe2x95x90C(Xa)xe2x80x94NRxe2x80x2xe2x80x94, xe2x80x94NRxe2x80x2xe2x80x94C(Xa)xe2x95x90Nxe2x80x94, xe2x80x94P(O)(ORxe2x80x2)xe2x80x94Oxe2x80x94, xe2x80x94Oxe2x80x94P(O)(ORxe2x80x2)xe2x80x94, xe2x80x94S(O)nCRxe2x80x2Rxe2x80x3xe2x80x94, xe2x80x94S(O)nxe2x80x94NRxe2x80x2xe2x80x94, xe2x80x94NRxe2x80x2xe2x80x94S(O)nxe2x80x94, and a covalent bond; where n is 0, 1 or 2; and R, Rxe2x80x2 and Rxe2x80x3 at each separate occurrence are selected from the group consisting of hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, alkynyl, substituted alkynyl, aryl, heteroaryl, and heterocyclic; and pharmaceutically acceptable salts, individual isomer, mixtures of isomers, and prodrugs thereof provided that the multibinding compound of Formula (I) cannot be:
(i) a compound of formula: 
where the linker, X, is xe2x80x94O-(alkylene)-Oxe2x80x94, xe2x80x94O-(hydroxyalkylene)-Oxe2x80x94, xe2x80x94Oxe2x80x94[(CH2)2- [(OCH2CH2)n]xe2x80x94Oxe2x80x94 (where n is an integer of 1 to 3), or xe2x80x94NHCO-(alkylene)-CONHxe2x80x94; and
(ii) a compound of formula RaRbNCH(CH3)CH2[xe2x80x94OCH2CH(CH3)xe2x80x94]2-8NRaRb wherein Ra is an aryl-OCH2CH(OH)CH2xe2x80x94 group and Rb is either hydrogen or an aryl-OCH2CH(OH)CH2xe2x80x94 group where the aryl group is 2-allylphenyl, 4-(2-methoxyethyl)phenyl, 1-naphthyl, or 4-methoxyphenyl.
Preferably, q is less than p in the multibinding compounds of this invention.
In another aspect, this invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and an effective amount of a multibinding compound of Formula (I): 
wherein:
p is an integer of from 2 to 10;
q is an integer of from 1 to 20,
each ligand, L, is independently of each other:
(i) a compound of formula (a): 
wherein:
Ar1 is selected from the group consisting of aryl, heteroaryl, cycloalkyl, substituted cycloalkyl, and heterocyclyl wherein each of said Ar1 substituent optionally links the ligand to a linker via a covalent bond;
R1 and R2 are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, and a covalent bond that links the ligand to a linker;
R3 is selected from the group consisting of hydrogen, alkyl, and a covalent bond that links the ligand to a linker; or
(ii) a compound of formula (b): 
wherein:
Ar2 is selected from the group consisting of aryl, heteroaryl, cycloalkyl, substituted cycloalkyl, and heterocyclyl wherein each of said Ar2 substituent optionally links the ligand to a linker via a covalent bond;
R4 and R5 are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, and a covalent bond that links the ligand to a linker;
W is a covalent bond, alkyl, or substituted alkyl where one or more of the carbon atoms in said alkyl or substituted alkyl group is optionally replaced by one or more heteroatom selected from xe2x80x94Oxe2x80x94, S(O)nxe2x80x94 (where n is an integer from 0 to 2), or xe2x80x94NR4xe2x80x94 (where R4 is hydrogen, or alkyl); and
Ar3 is selected from the group consisting of aryl, heteroaryl, cycloalkyl, substituted cycloalkyl, and heterocycle wherein each of said Ar3 substituent optionally links the ligand to a linker via a covalent bond;
each linker, X, in the multibinding compound of Formula (I) independently has the formula:
xe2x80x94Xaxe2x80x94Zxe2x80x94(Yaxe2x80x94Z)mxe2x80x94Xaxe2x80x94
xe2x80x83wherein:
m is an integer of from 0 to 20;
Xa at each separate occurrence is selected from the group consisting of xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94NRxe2x80x94, xe2x80x94C(O)xe2x80x94, xe2x80x94C(O)Oxe2x80x94, xe2x80x94OC(O)xe2x80x94, xe2x80x94C(O)NRxe2x80x94, xe2x80x94NRC(O)xe2x80x94, C(S), xe2x80x94C(S)Oxe2x80x94, xe2x80x94C(S)NRxe2x80x94, xe2x80x94NRC(S)xe2x80x94, or a covalent bond where R is as defined below;
Z at each separate occurrence is selected from the group consisting of alkylene, substituted alkylene, cycloalkylene, substituted cylcoalkylene, alkenylene, substituted alkenylene, alkynylene, substituted alkynylene, cycloalkenylene, substituted cycloalkenylene, arylene, heteroarylene, heterocyclene, or a covalent bond;
each Ya at each separate occurrence is selected from the group consisting of xe2x80x94Oxe2x80x94, xe2x80x94C(O)xe2x80x94, xe2x80x94OC(O)xe2x80x94, xe2x80x94C(O)Oxe2x80x94, xe2x80x94NRxe2x80x94, xe2x80x94S(O)nxe2x80x94, xe2x80x94C(O)NRxe2x80x2xe2x80x94, xe2x80x94NRxe2x80x2C(O)xe2x80x94, xe2x80x94NRxe2x80x2C(O)NRxe2x80x2xe2x80x94, xe2x80x94NRxe2x80x2C(S)NRxe2x80x2xe2x80x94, xe2x80x94C(xe2x95x90NRxe2x80x2)xe2x80x94NRxe2x80x2xe2x80x94, xe2x80x94NRxe2x80x2xe2x80x94C(xe2x95x90NRxe2x80x2)xe2x80x94, xe2x80x94OC(O)xe2x80x94NRxe2x80x2xe2x80x94, xe2x80x94NRxe2x80x2xe2x80x94C(O)xe2x80x94Oxe2x80x94, Nxe2x95x90C(Xa)xe2x80x94NRxe2x80x2xe2x80x94, xe2x80x94NRxe2x80x2xe2x80x94C(Xa)xe2x95x90Nxe2x80x94,xe2x80x94P(O)(ORxe2x80x2)xe2x80x94Oxe2x80x94, xe2x80x94Oxe2x80x94P(O)(ORxe2x80x2)xe2x80x94, xe2x80x94S(O)nCRxe2x80x2Rxe2x80x3xe2x80x94, xe2x80x94S(O)nxe2x80x94NRxe2x80x2xe2x80x94, xe2x80x94NRxe2x80x2xe2x80x94S(O)nxe2x80x94, and a covalent bond; where n is 0, 1 or 2; and R, Rxe2x80x2 and Rxe2x80x3 at each separate occurrence are selected from the group consisting of hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, alkynyl, substituted alkynyl, aryl, heteroaryl, and heterocyclic; and pharmaceutically acceptable salts, individual isomer, mixtures of isomers, and prodrugs thereof provided that the multibinding compound of Formula (I) cannot be:
(i) a compound of formula: 
xe2x80x83where the linker, X, is xe2x80x94O-(alkylene)-Oxe2x80x94, xe2x80x94O-(hydroxyalkylene)-Oxe2x80x94, xe2x80x94Oxe2x80x94[(CH2)2- [(OCH2CH2)n]xe2x80x94Oxe2x80x94 (where n is an integer of 1 to 3), or xe2x80x94NHCO-(alkylene)-CONHxe2x80x94; and
(ii) a compound of formula RaRbNCH(CH3)CH2[xe2x80x94OCH2CH(CH3)xe2x80x94]2-8NRaRb wherein Ra is an aryl-OCH2CH(OH)CH2xe2x80x94 group and Rb is either hydrogen or an aryl-OCH2CH(OH)CH2xe2x80x94 group where the aryl group is 2-allylphenyl, 4-(2-methoxyethyl)phenyl, 1-naphthyl, or 4-methoxyphenyl.
In still another aspect, this invention provides a method of treating diseases mediated by a xcex23 adrenergic receptor in a mammal, said method comprising administering to said mammal a therapeutically effective amount of a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a multibinding compound of Formula (I): 
wherein:
p is an integer of from 2 to 10;
q is an integer of from 1 to 20,
each ligand, L, is independently of each other:
(i) a compound of formula (a): 
wherein:
Ar1 is selected from the group consisting of aryl, heteroaryl, cycloalkyl, substituted cycloalkyl, and heterocyclyl wherein each of said Ar1 substituent optionally links the ligand to a linker via a covalent bond;
R1 and R2 are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, and a covalent bond that links the ligand to a linker;
R3 is selected from the group consisting of hydrogen, alkyl, and a covalent bond that links the ligand to a linker; or
(ii) a compound of formula (b): 
wherein:
Ar2 is selected from the group consisting of aryl, heteroaryl, cycloalkyl, substituted cycloalkyl, and heterocyclyl wherein each of said Ar2 substituent optionally links the ligand to a linker via a covalent bond;
R4 and R5 are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, and a covalent bond that links the ligand to a linker;
W is a covalent bond, alkyl, or substituted alkyl where one or more of the carbon atoms in said alkyl or substituted alkyl group is optionally replaced by one or more heteroatom selected from xe2x80x94Oxe2x80x94, S(O)nxe2x80x94 (where n is an integer from 0 to 2), or xe2x80x94NR4xe2x80x94 (where R4 is hydrogen, or alkyl); and
Ar3 is selected from the group consisting of aryl, heteroaryl, cycloalkyl, substituted cycloalkyl, and heterocycle wherein each of said Ar3 substituent optionally links the ligand to a linker via a covalent bond;
each linker, X, in the multibinding compound of Formula (I) independently has the formula:
xe2x80x94Xaxe2x80x94Zxe2x80x94(Yaxe2x80x94Z)mxe2x80x94Xaxe2x80x94
xe2x80x83wherein:
m is an integer of from 0 to 20;
Xa at each separate occurrence is selected from the group consisting of xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94NRxe2x80x94, xe2x80x94C(O)xe2x80x94, xe2x80x94C(O)Oxe2x80x94, xe2x80x94OC(O)xe2x80x94, xe2x80x94C(O)NRxe2x80x94, xe2x80x94NRC(O)xe2x80x94, C(S), xe2x80x94C(S)Oxe2x80x94, xe2x80x94C(S)NRxe2x80x94, xe2x80x94NRC(S)xe2x80x94, or a covalent bond where R is as defined below;
Z at each separate occurrence is selected from the group consisting of alkylene, substituted alkylene, cycloalkylene, substituted cylcoalkylene, alkenylene, substituted alkenylene, alkynylene, substituted alkynylene, cycloalkenylene, substituted cycloalkenylene, arylene, heteroarylene, heterocyclene, or a covalent bond;
each Ya at each separate occurrence is selected from the group consisting of xe2x80x94Oxe2x80x94, xe2x80x94C(O)xe2x80x94, xe2x80x94OC(O)xe2x80x94, xe2x80x94C(O)Oxe2x80x94, xe2x80x94NRxe2x80x94, xe2x80x94S(O)nxe2x80x94, xe2x80x94C(O)NRxe2x80x2xe2x80x94, xe2x80x94NRxc2x0C(O)xe2x80x94, xe2x80x94NRxe2x80x2C(O)NRxe2x80x2xe2x80x94, xe2x80x94NRxe2x80x2C(S)NRxe2x80x2xe2x80x94, xe2x80x94C(xe2x95x90NRxe2x80x2)xe2x80x94NRxe2x80x2xe2x80x94, xe2x80x94NRxe2x80x2xe2x80x94C(xe2x95x90NRxe2x80x2)xe2x80x94, xe2x80x94OC(O)xe2x80x94NRxe2x80x2xe2x80x94, xe2x80x94NRxe2x80x2xe2x80x94C(O)xe2x80x94Oxe2x80x94, xe2x80x94Nxe2x95x90C(Xa)xe2x80x94NRxe2x80x2xe2x80x94, xe2x80x94NRxe2x80x2xe2x80x94C(Xa)xe2x95x90Nxe2x80x94,xe2x80x94P(O)(ORxe2x80x2)xe2x80x94Oxe2x80x94, xe2x80x94Oxe2x80x94P(O)(ORxe2x80x2)xe2x80x94, xe2x80x94S(O)nCRxe2x80x2Rxe2x80x3xe2x80x94, xe2x80x94S(O)nxe2x80x94NRxe2x80x2xe2x80x94, xe2x80x94NRxe2x80x2xe2x80x94S(O)nxe2x80x94, and a covalent bond; where n is 0, 1 or 2; and R, Rxe2x80x2 and Rxe2x80x3 at each separate occurrence are selected from the group consisting of hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, alkynyl, substituted alkynyl, aryl, heteroaryl, and heterocyclic; and pharnaceutically acceptable salts, individual isomer, mixtures of isomers, and prodrugs thereof provided that the multibinding compound of Formula (I) cannot be:
(i) a compound of formula: 
xe2x80x83where the linker, X, is xe2x80x94O-(alkylene)-Oxe2x80x94, xe2x80x94O-(hydroxyalkylene)-Oxe2x80x94, xe2x80x94Oxe2x80x94[(CH2)2- [(OCH2CH2)n]xe2x80x94Oxe2x80x94 (where n is an integer of 1 to 3), or xe2x80x94NHCO-(alkylene)-CONHxe2x80x94; and
(ii) a compound of formula RaRbNCH(CH3)CH2[xe2x80x94OCH2CH(CH3)xe2x80x94]2-8NRaRb wherein Ra is an aryl-OCH2CH(OH)CH2xe2x80x94 group and Rb is either hydrogen or an aryl-OCH2CH(OH)CH2xe2x80x94 group where the aryl group is 2-allylphenyl, 4-(2-methoxyethyl)phenyl, 1-naphthyl, or 4-methoxyphenyl.
In still another aspect, this invention is directed to general synthetic methods for generating large libraries of diverse multimeric compounds which multimeric compounds are candidates for possessing multibinding properties for xcex23 adrenergic receptor. The diverse multimeric compound libraries provided by this invention are synthesized by combining a linker or linkers with a ligand or ligands to provide for a library of multimeric compounds wherein the linker and ligand each have complementary functional groups permitting covalent linkage. The library of linkers is preferably selected to have diverse properties such as valency, linker length, linker geometry and rigidity, hydrophilicity or hydrophobicity, amphiphilicity, acidity, basicity and polarization. The library of ligands is preferably selected to have diverse attachment points on the same ligand, different functional groups at the same site of otherwise the same ligand, and the like.
This invention is also directed to libraries of diverse multimeric compounds which multimeric compounds are candidates for possessing multibinding properties for xcex23 adrenergic receptor. These libraries are prepared via the methods described above and permit the rapid and efficient evaluation of what molecular constraints impart multibinding properties to a ligand or a class of ligands targeting a receptor.
Accordingly, in one of its method aspects, this invention is directed to a method for identifying multimeric ligand compounds possessing multibinding properties for xcex23 adrenergic receptor which method comprises:
(a) identifying a ligand or a mixture of ligands wherein each ligand contains at least one reactive functionality;
(b) identifying a library of linkers wherein each linker in said library comprises at least two functional groups having complementary reactivity to at least one of the reactive functional groups of the ligand;
(c) preparing a multimeric ligand compound library by combining at least two stoichiometric equivalents of the ligand or mixture of ligands identified in (a) with the library of linkers identified in (b) under conditions wherein the complementary functional groups react to form a covalent linkage between said linker and at least two of said ligands; and
(d) assaying the multimeric ligand compounds produced in (c) above to identify multimeric ligand compounds possessing multibinding properties for xcex23 adrenergic receptor.
In another of its method aspects, this invention is directed to a method for identifying multimeric ligand compounds possessing multibinding properties for xcex23 adrenergic receptor which method comprises:
(a) identifying a library of ligands wherein each ligand contains at least one reactive functionality;
(b) identifying a linker or mixture of linkers wherein each linker comprises at least two functional groups having complementary reactivity to at least one of the reactive functional groups of the ligand;
(c) preparing a multimeric ligand compound library by combining at least two stoichiometric equivalents of the library of ligands identified in (a) with the linker or mixture of linkers identified in (b) under conditions wherein the complementary functional groups react to form a covalent linkage between said linker and at least two of said ligands; and
(d) assaying the multimeric ligand compounds produced in (c) above to identify multimeric ligand compounds possessing multibinding properties for xcex23 adrenergic receptor.
The preparation of the multimeric ligand compound library is achieved by either the sequential or concurrent combination of the two or more stoichiometric equivalents of the ligands identified in (a) with the linkers identified in (b). Sequential addition is preferred when a mixture of different ligands is employed to ensure heterodimeric or multimeric compounds are prepared. Concurrent addition of the ligands occurs when at least a portion of the multimer comounds prepared are homomultimeric compounds.
The assay protocols recited in (d) can be conducted on the multimeric ligand compound library produced in (c) above, or preferably, each member of the library is isolated by preparative liquid chromatography mass spectrometry (LCMS).
In one of its composition aspects, this invention is directed to a library of multimeric ligand compounds which may possess multivalent properties for xcex23 adrenergic receptor which library is prepared by the method comprising:
(a) identifying a ligand or a mixture of ligands wherein each ligand contains at least one reactive functionality;
(b) identifying a library of linkers wherein each linker in said library comprises at least two functional groups having complementary reactivity to at least one of the reactive functional groups of the ligand; and
(c) preparing a multimeric ligand compound library by combining at least two stoichiometric equivalents of the ligand or mixture of ligands identified in (a) with the library of linkers identified in (b) under conditions wherein the complementary functional groups react to form a covalent linkage between said linker and at least two of said ligands.
In another of its composition aspects, this invention is directed to a library of multimeric ligand compounds which may possess multivalent properties for xcex23 adrenergic receptor which library is prepared by the method comprising:
(a) identifying a library of ligands wherein each ligand contains at least one reactive functionality;
(b) identifying a linker or mixture of linkers wherein each linker comprises at least two functional groups having complementary reactivity to at least one of the reactive functional groups of the ligand; and
(c) preparing a multimeric ligand compound library by combining at least two stoichiometric equivalents of the library of ligands identified in (a) with the linker or mixture of linkers identified in (b) under conditions wherein the complementary functional groups react to form a covalent linkage between said linker and at least two of said ligands.
In a preferred embodiment, the library of linkers employed in either the methods or the library aspects of this invention is selected from the group comprising flexible linkers, rigid linkers, hydrophobic linkers, hydrophilic linkers, linkers of different geometry, acidic linkers, basic linkers, linkers of different polarization and amphiphilic linkers. For example, in one embodiment, each of the linkers in the linker library may comprise linkers of different chain length and/or having different complementary reactive groups. Such linker lengths can preferably range from about 2 to 100 xc3x85.
In another preferred embodiment, the ligand or mixture of ligands is selected to have reactive functionality at different sites on said ligands in order to provide for a range of orientations of said ligand on said multimeric ligand compounds. Such reactive functionality includes, by way of example, carboxylic acids, carboxylic acid halides, carboxyl esters, amines, halides,pseudohalides, isocyanates, vinyl unsaturation, ketones, aldehydes, thiols, alcohols, anhydrides, boronates, and precursors thereof. It is understood, of course, that the reactive functionality on the ligand is selected to be complementary to at least one of the reactive groups on the linker so that a covalent linkage can be formed between the linker and the ligand.
In other embodiments, the multimeric ligand compound is homomeric (i.e., each of the ligands is the same, although it may be attached at different points) or heteromeric (i.e., at least one of the ligands is different from the other ligands).
In addition to the combinatorial methods described herein, this invention provides for an iterative process for rationally evaluating what molecular constraints impart multibinding properties to a class of multimeric compounds or ligands targeting a receptor. Specifically, this method aspect is directed to a method for identifying multimeric ligand compounds possessing multibinding properties for xcex23 adrenergic receptor which method comprises:
(a) preparing a first collection or iteration of multimeric compounds which is prepared by contacting at least two stoichiometric equivalents of the ligand or mixture of ligands which target a receptor with a linker or mixture of linkers wherein said ligand or mixture of ligands comprises at least one reactive functionality and said linker or mixture of linkers comprises at least two functional groups having complementary reactivity to at least one of the reactive functional groups of the ligand wherein said contacting is conducted under conditions wherein the complementary functional groups react to form a covalent linkage between said linker and at least two of said ligands;
(b) assaying said first collection or iteration of multimeric compounds to assess which if any of said multimeric compounds possess multibinding properties for xcex23 adrenergic receptor;
(c) repeating the process of (a) and (b) above until at least one multimeric compound is found to possess multibinding properties for xcex23 adrenergic receptor;
(d) evaluating what molecular constraints imparted multibinding properties to the multimeric compound or compounds for xcex23 adrenergic receptor found in the first iteration recited in (a)-(c) above;
(e) creating a second collection or iteration of multimeric compounds which elaborates upon the particular molecular constraints imparting multibinding properties to the multimeric compound or compounds found in said first iteration;
(f) evaluating what molecular constraints imparted enhanced multibinding properties to the multimeric compound or compounds found in the second collection or iteration recited in (e) above;
(g) optionally repeating steps (e) and (f) to further elaborate upon said molecular constraints.
Preferably, steps (e) and (f) are repeated at least two times, more preferably at from 2-50 times, even more preferably from 3 to 50 times, and still more preferably at least 5-50 times.